US5817755A - Synthetic peptide analogs of cross-linked N-telopeptides of type I collagen - Google Patents
Synthetic peptide analogs of cross-linked N-telopeptides of type I collagen Download PDFInfo
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- US5817755A US5817755A US08/807,030 US80703097A US5817755A US 5817755 A US5817755 A US 5817755A US 80703097 A US80703097 A US 80703097A US 5817755 A US5817755 A US 5817755A
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 136
- 108010049937 collagen type I trimeric cross-linked peptide Proteins 0.000 title abstract description 7
- 108010022452 Collagen Type I Proteins 0.000 title abstract description 5
- 102000012422 Collagen Type I Human genes 0.000 title abstract description 5
- 235000001014 amino acid Nutrition 0.000 claims description 165
- 150000001413 amino acids Chemical class 0.000 claims description 163
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 claims description 117
- 125000000539 amino acid group Chemical group 0.000 claims description 22
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 19
- XVOYSCVBGLVSOL-UHFFFAOYSA-N cysteic acid Chemical compound OC(=O)C(N)CS(O)(=O)=O XVOYSCVBGLVSOL-UHFFFAOYSA-N 0.000 claims description 13
- 235000008206 alpha-amino acids Nutrition 0.000 claims description 11
- OYIFNHCXNCRBQI-UHFFFAOYSA-N 2-aminoadipic acid Chemical compound OC(=O)C(N)CCCC(O)=O OYIFNHCXNCRBQI-UHFFFAOYSA-N 0.000 claims description 10
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 9
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 9
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 9
- 229940079889 pyrrolidonecarboxylic acid Drugs 0.000 claims description 9
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 8
- 235000018417 cysteine Nutrition 0.000 claims description 7
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 7
- 239000002198 insoluble material Substances 0.000 claims description 7
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 claims description 4
- 150000001370 alpha-amino acid derivatives Chemical group 0.000 claims description 2
- 235000018102 proteins Nutrition 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims description 2
- 101150108015 STR6 gene Proteins 0.000 claims 1
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 22
- 230000003278 mimic effect Effects 0.000 abstract description 3
- 239000012634 fragment Substances 0.000 description 74
- ODHCTXKNWHHXJC-GSVOUGTGSA-N Pyroglutamic acid Natural products OC(=O)[C@H]1CCC(=O)N1 ODHCTXKNWHHXJC-GSVOUGTGSA-N 0.000 description 72
- ODHCTXKNWHHXJC-UHFFFAOYSA-N acide pyroglutamique Natural products OC(=O)C1CCC(=O)N1 ODHCTXKNWHHXJC-UHFFFAOYSA-N 0.000 description 72
- BEBCJVAWIBVWNZ-UHFFFAOYSA-N glycinamide Chemical compound NCC(N)=O BEBCJVAWIBVWNZ-UHFFFAOYSA-N 0.000 description 67
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 10
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 10
- SNDPXSYFESPGGJ-UHFFFAOYSA-N 2-aminopentanoic acid Chemical compound CCCC(N)C(O)=O SNDPXSYFESPGGJ-UHFFFAOYSA-N 0.000 description 8
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 8
- 125000003275 alpha amino acid group Chemical group 0.000 description 8
- 238000006467 substitution reaction Methods 0.000 description 7
- 239000004472 Lysine Substances 0.000 description 6
- 125000003277 amino group Chemical group 0.000 description 6
- SNDPXSYFESPGGJ-BYPYZUCNSA-N L-2-aminopentanoic acid Chemical compound CCC[C@H](N)C(O)=O SNDPXSYFESPGGJ-BYPYZUCNSA-N 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 5
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 4
- 150000001371 alpha-amino acids Chemical class 0.000 description 4
- 238000003018 immunoassay Methods 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 230000008033 biological extinction Effects 0.000 description 3
- 210000000988 bone and bone Anatomy 0.000 description 3
- 229920001436 collagen Polymers 0.000 description 3
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 3
- 239000002207 metabolite Substances 0.000 description 3
- 210000002700 urine Anatomy 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- CNMAQBJBWQQZFZ-LURJTMIESA-N (2s)-2-(pyridin-2-ylamino)propanoic acid Chemical compound OC(=O)[C@H](C)NC1=CC=CC=N1 CNMAQBJBWQQZFZ-LURJTMIESA-N 0.000 description 2
- 208000006386 Bone Resorption Diseases 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- -1 Leu Chemical group 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 2
- 230000024279 bone resorption Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000004816 latex Substances 0.000 description 2
- 229920000126 latex Polymers 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- PKEXHDRGMPOQQN-UHFFFAOYSA-N 2-aminohexanedioic acid Chemical group OC(=O)C(N)CCCC(O)=O.OC(=O)C(N)CCCC(O)=O PKEXHDRGMPOQQN-UHFFFAOYSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 101710125089 Bindin Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- ZAHDXEIQWWLQQL-IHRRRGAJSA-N Deoxypyridinoline Chemical compound OC(=O)[C@@H](N)CCCC[N+]1=CC(O)=C(C[C@H](N)C([O-])=O)C(CC[C@H](N)C(O)=O)=C1 ZAHDXEIQWWLQQL-IHRRRGAJSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical group O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010048038 N-propeptide type I collagen Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 1
- LCYXYLLJXMAEMT-SAXRGWBVSA-N Pyridinoline Chemical compound OC(=O)[C@@H](N)CCC1=C[N+](C[C@H](O)CC[C@H](N)C([O-])=O)=CC(O)=C1C[C@H](N)C(O)=O LCYXYLLJXMAEMT-SAXRGWBVSA-N 0.000 description 1
- 101100386054 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) CYS3 gene Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 230000026045 iodination Effects 0.000 description 1
- 238000006192 iodination reaction Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- NYCVCXMSZNOGDH-UHFFFAOYSA-N pyrrolidine-1-carboxylic acid Chemical compound OC(=O)N1CCCC1 NYCVCXMSZNOGDH-UHFFFAOYSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 101150035983 str1 gene Proteins 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 150000003573 thiols Chemical group 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
Definitions
- This invention relates to immunoassays and provides a synthetic peptide that acts as an immunoreactive analog of the natural cross-linked amino-terminal telopeptide of type I collagen.
- N-telopeptides of type I collagen are excreted in urine as end-products of the process of bone resorption. These metabolites can be measured by immunoassay to provide an accurate and specific index of bone resorption activity, for example, using the monoclonal antibody (mAb) 1H11 produced by hybridoma 1H11 (ATCC No. HB 10611).
- the epitope recognized by mAb 1H11 is embodied in forms of the excreted cross-linked N-telopeptides that include the collagen ⁇ 2(I) N-telopeptide sequence Gln-Tyr-Asp-Gly-K-Gly-Val-Gly, together with a linked second telopeptide from either ⁇ 1(I) or ⁇ 2(I), wherein K is embodied in the cross-link.
- NTx metabolite excreted in urine is shown: ##STR1## wherein ##STR2## is hydroxylysyl pyridinoline or lysyl pyridinoline, and Gln is glutamine (Q) or wholly cyclized pyrrolidone carboxylic acid (5-oxo-2-pyrrolidinebarboxylic acid, i.e., pyroglutamic acid) (J).
- Linear peptides synthesized to match the human ⁇ 1(l) or ⁇ 2(l) N-telopeptide sequences, and in which K is simply lysine, are respectively not recognized or recognized very weakly by mAb 1H11.
- the hybridoma cell line that produces the mAb 1H11 was deposited on Nov. 20, 1990, at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, USA, under accession number HB 10611.
- the subject peptides are embodied by: Gln-Tyr-Xaa-Gly-Xaa-Gly-Val-Gly, wherein Gln is pyrrolidone carboxylic acid, wherein residue Xaa in position 2 is Asp, Glu, or other ⁇ -amino acid with a negatively charged side chain (R group), and wherein the structure of residue Xaa in position 5 is such that the peptide mimics features of the cross-linked NTx conformation and chemistry such that the peptide binds to mAb 1H11.
- Xaa in position 5 is selected from among Asp, Glu, and other negatively charged amino acids.
- Xaa in position 5 is selected from among Pro, Nva (norvaline), Trp, His, and conservative substitutions therefor that provide the requisite degree of binding to mAb 1H11.
- Xaa in position 5 bears a thiol group, in which case the peptides may dimerize via a cystine bridge.
- Xaa in position 5 bears an amino or thiol group that is conjugated to a carrier molecule or insoluble material.
- the invention provides synthetic linear peptides which mimic the epitope recognized by mAb 1H11 in cross-linked NTx.
- the subject peptides are embodied by the generic formula Gln-Tyr-Xaa-Gly-Xaa-Gly-Val-Gly, wherein Gln is pyrolidone carboxylic acid, residue Xaa in position 3 is negatively charged, and the structure of residue Xaa in position 5 is such that the peptide mimics the NTx conformation and chemistry to the extent that the peptide binds to mAb 1H11.
- Representative peptides designated by SEQ ID NOS: 1-59 are shown in the Table below, along with their relative binding affinities for mAb 1H11.
- the peptide Gln-Tyr-Asp-Gly-Glu-Gly-Val-Gly had a binding affinity for mAb 1H11 comparable to that of the cross-linked NTx antigen.
- the mAb 1H11 inhibition curves for NTx (derived from either urine or bone) and Gln-Tyr-Asp-Gly-Glu-Gly-Val-Gly are essentially parallel, indicating that the synthetic peptide mimics the natural epitope.
- Such preferred embodiments of the subject synthetic peptides bind to mAb 1H11 at a substantially equimolar extent as does the cross-linked NTx metabolite (shown above) in antibody inhibition assays.
- Gln-Tyr-Asp-Gly-Glu-Gly-Val-Gly represents the requisite size for presenting the epitope recognized by mAb 1H11.
- the degrees to which individual amino acids in the core sequence are considered essential for mAb 1H11 epitope binding are as follows. Binding to mAb 1H11 occurs when the N-terminal Gln (position 1) is in the cyclized form, pyrrolidine carboxylic acid (J), as occurs naturally in collagen chains.
- the core sequence can be formed with pyrrolidone carboxylic acid in position 1 by initially sequencing the core sequence with Glu in position 1, followed by autoclaving the peptide. Autoclaving causes the cyclization of Glu to pyrrolidone carboxylic acid.
- the Tyr residue in position 2 is an essential feature, as iodination of Tyr or substitution with Phe (SEQ ID NO:36) in this position blocks mAb 1H11 binding.
- the Asp residue in position 3 is also considered important, as substituents that eliminate the negative charge at this position block mAb 1H11 binding (based upon binding studies with NTx of modified structure from animal species other than human).
- the Asp in position 3 may be replaced by Glu (SEQ ID NO:30) or other ⁇ -amino acid residue bearing a negatively charged R group.
- the Gly residue in position 4 is important, as the conversative substitution of either Val (SEQ ID NO:37) or Ala (SEQ ID NO:27) at this position substantially eliminates mAb 1H11 binding affinity.
- Gly residues at positions 6 and 8 are equally important, as various substitutions at these positions, including Ala, Val, Leu, Pro, and Cys, lead to loss of mAb 1H11 binding affinity. (See SEQ ID NOS:28, 35, 46, 49, 52, 54, and 56.)
- Val residue at position 7 is important, as conservative substitutions of other hydrophobic substituents, such as Leu (SEQ ID NO:29) or Ile (SEQ ID NO:55), eliminate the requisite binding of mAb 1H11.
- the invention provides, in a first embodiment, a peptide that binds to mAb 1H11, comprising Gln-Tyr-Xaa-Gly-Xaa-Gly-Val-Gly (SEQ ID NO:60), wherein Gln is pyrrolidone carboxylic acid, Xaa in position 3 is a negatively charged amino acid residue, and Xaa in position 5 is also an amino acid with a negatively charged R group.
- a suitable amino acid with a negatively charged R group is selected from among Glu, Asp, and other natural and synthetic a-amino acids which bear a negatively charged R group such as a carboxyl, sulfate, or phosphate group. Representative examples include ⁇ -aminoadipic acid (SEQ ID NO:34) and cysteic acid (SEQ ID NO:26).
- the Xaa residue in position 5 is selected from among Pro, Trp, Nva, and His (SEQ ID NO:61) or conservative substitutions therefor that afford a requisite degree of mAb 1H11 binding.
- Representative amino acids with a nonpolar R group include Pro (SEQ ID NO: 13), Trp (SEQ ID NO: 19), norvaline (SEQ ID NO:38), and other natural and synthetic ⁇ -amino acids which bear a nonpolar R group such as characterize Ala, Ile, Leu, Met, Phe, Pro, Trp, and Val.
- Cys in position 5 (SEQ ID NO:2) provided excellent binding to mAb 1H11. It is contemplated that other natural and synthetic ⁇ -amino acids which bear a polar but uncharged R group such as characterize Asn, Cys, Gln, Gly, Ser, Thr, and Tyr may also be suitable. Representative examples include pyridinylalanine (SEQ ID NO:62) (Bachem) and other ⁇ -amino acids wherein the R group has a pyridine ring. Cysteine and other thiol bearing ⁇ -amino acids are particularly preferred embodiments in terms of the degree of mAb 1H11 binding.
- the invention provides a peptide that binds to mAb 1H11, comprising Gln-Tyr-Xaa-Gly-Xaa-Gly-Val-Gly (SEQ ID NO:63), wherein Gin is pyrrolidone carboxylic acid, Xaa in position 3 is a negatively charged amino acid residue, and Xaa in position 5 is an ⁇ -amino acid bearing a thiol group.
- Representative examples include cysteine (SEQ ID NO:2), homocysteine (SEQ ID NO:64), and related residues in which R contains 0-3 carbon atoms.
- each of the residues Xaa in position 5 may be the same or a different ⁇ -amino acid bearing a side chain (R) with a thiol group.
- the natural epitope embodied in urinary NTx is mimicked by conjugating the synthetic peptide Gln-Tyr-Xaa-Gly-Xaa-Gly-Val-Gly (SEQ ID NO:65) to either a solute or an insoluble material.
- the Gln and the Xaa in position 3 are as defined above, and the Xaa in position 5 is an ⁇ -amino acid bearing a side chain amino or thiol group.
- Xaa in position 5 may be lysine (SEQ ID NO:9) with an epsilon amino group, in which case conjugation to the solute or insoluble material is through the amino group, for example by glutaraldehyde using standard procedures.
- Representative Xaa residues for this purpose are lysine and cysteine (SEQ ID NO:2), and representative linking agents, for conjugation to, e.g., bovine serum albumin, are glutaraldehyde and m-malieimidobenzoyl-N-hydroxy-succinimide ester (MBS), respectively.
- solute is meant a molecular substance dissolved in aqueous solution.
- Representive solutes for this purpose include proteins, such as keyhole limpet hemocyanin (KLH), albumin, and enzymes, peptides such as Gln-Tyr-Asp-Gly-Cys-Gly-Val-Gly (or Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly), and other soluble molecules such as biotin, avidin, and fluorescent and chemiluminescent moieties.
- Representative insoluble materials include latex particles, dipsticks, micro-titer wells, and other substrata used in heterogeneous immunoassays.
- the antibody-binding affinity of the Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly peptide for mAb 1H11 is increased about 50-fold, approximating the binding affinity of the natural epitope.
- a conjugate can be used in solution as a competing antigen or as a coating agent for solid surfaces, for example, to coat wells in a microtiter-plate kit or latex or other particles in automated assay systems.
- this fourth embodiment of the invention provides a conjugated peptide that binds to mAb 1H11, comprising: ##STR4## wherein Gln is pyrrolidone carboxylic acid, Xaa in position 3 is a negatively charged amino acid residue, Xaa in position 5 is an amino acid residue bearing an amino group or thiol group, and R is either a solute or an insoluble material conjugated through the amino group or thiol group to residue Xaa in position 5.
- the synthetic peptides of the present invention are prepared by methods well known in the art, for example as summarized in A. P. Gloor, et al., Synthesis Notes, Novabiochem Catalog and Peptide Synthesis Handbook, La Jolla, Calif., pp. S1-S5, 1994-1995.
- synthetic peptides were purchased from an outside vendor, or made in house using the Symphony/Multiplex (TM) multiple peptide synthesizer from Rainin Instrument Co. (Woburn, Mass.)
- a milligram per mL solution of the peptide was made in PBS.
- the absorbence at A280 was read and the concentration confirmed.
- Dilutions of the peptide were then made in a buffer such as OSTEOMARK (TM) calibrator diluent (OSTEOMARK Assay, Ostex International, Seattle, Wash.).
- Working strength concentrations were between 10 and 0.01 micrograms per milliliter.
- OSTEOMARK assay was run using OSTEOMARK kits and components, including monoclonal antibody 1H11.
- a standard curve was run using OSTEOMARK calibrators. Peptide samples were generally tested at 10, 3, 1, and down to 0.01 ⁇ G per mL.
- the standard OSTEOMARK curve was generated using a four parameter logistic curve fit program (MedData Inc., N.Y., N.Y.). Peptide values were back fit into the standard curve and values were reported in picomoles Bone Collagen Equivalents (BCE)/mL (which equals nanomolar BCE, or nM BCE). As not all peptides that competed for 1H11 showed a linear response, values between 0.5 and 1 optical density units were taken and averaged.
- BCE Bone Collagen Equivalents
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Abstract
Description
TABLE ______________________________________ Synthetic Linear Peptides Alpha 2 chain of type 1 collagen SEQ Amino acid position number ID NO: 0 1 2 3 4 5 6 7 8 9 10 11 Results.sup.1 ______________________________________ 1 J Y D G A G V G 483 2 J Y D G C G V G 2500 3 J Y D G D G V G 1840 4 J Y D G E G V G 2400 5 J Y D G F G V G 365 6 J Y D G G G V G 114 7 J Y D G H G V G 650 8 J Y D G I G V G 250 9 J Y D G K G V G 100 10 J Y D G L G V G 230 11 J Y D G M G V G 183 12 J Y D G N G V G 135 13 J Y D G P G V G 2056 14 J Y D G Q G V G 148 15 J Y D G R G V G 23 16 J Y D G S G V G 30 17 J Y D G T G V G 252 18 J Y D G V G V G 400 19 J Y D G W G V G 1300 20 J Y D G Y G V G 104 21 J Y D G R G V A 6 22 J Y D G E G V L <50 23 J Y D G E G V <50 24 J Y D G E <50 25 Y D G E G V <50 26 j Y D G X.sup.2 G V G 1000 27 J Y D A E G V G <50 28 J Y D G E A V G <50 29 J Y D G E G V G <50 30 J Y E G E G V G 3000 31 J Y D G D G V G 1400 32 J Y E G D G V G 1000 33 E.sup.3 Y D G E G V G 2000 34 J Y D G X.sup.4 G V G 750 35 J Y D G E G V V 5 36 J F D G E G V G 14 37 J Y D V E G V G 6 38 J Y D G X.sup.5 G V G 860 39 Q Y D G E G V G <50 40 J Y D G E G V G L <50 41 G G Y D G E G V G <50 42 J Y D G E G V G G K L <50 43 S Y D G E G V G <50 44 J Y D G E G V X.sup.6 <50 45 G V G C G D Y Q <50 46 J Y D G E V V G <50 47 J Y D G E G G G <50 48 J Y D G E G A G <50 49 J Y D G E G V A <50 50 H Y D G E G V G nd 51 P Y D G E G V G <50 52 J Y D G E C G G <50 53 J V G E G D Y N <50 54 J Y D G E G V P <50 55 J Y D G E G I G <50 56 J Y D G E G V C <50 57 X.sup.7 Y D G E G V G nd.sup.8 58 J Y D G X.sup.9 G V G 135 59 J Y D G X.sup.10 G V G nd ______________________________________ .sup.1 Peptide binding to mAb 1H11 is expressed in the following units: 1μg synthetic peptide = X nmole BCE, where X is the value in the Table For comparison, the native sequence, JYDGKGVG (SEQ ID NO:9), gave an X value of 100 in this assay. .sup.2 X = cysteic acid (2amino-3-sulfopropanoic acid). That is, cysteine with a sulfite (--SO.sub.3.sup.-2) group substituted for the normal mercapto (--SH) group, preventing disulfide bridges. .sup.3 The sequence EYDGEGVG is not recognized by 1H11. Here, by autoclaving this peptide under conditions that convert the glutamic acid in position 1 into pyroglutamic acid, substantial increase in 1H11 bindin resulted. Therefore, the N terminus pyroglutamic acid is part of the epitope. .sup.4 X = aminoadipic acid (2amino hexanedioic acid), in which the side chain amino group of lysine is replaced by a negatively charged carboxyl group. .sup.5 X = Norvaline. .sup.6 X = G--NH.sub.2. The carboxyl group of the Gly residue in position 8 is normally exposed (not in peptide bond). Here, an amino group was added to the carboxyl group (to make 1,2diaminoacetaldehyde), which prevented recognition. Therefore, the free carboxyl group is considered part of the epitope. .sup.7 X = 4hydroxyproline. .sup.8 nd = not determined. .sup.9 X = Biotinolated lysine (K). .sup.10 X = Biotinolated cysteine (C).
__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 74 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: XaaTyrAspGlyAlaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: XaaTyrAspGlyCysGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: XaaTyrAspGlyAspGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: XaaTyrAspGlyGluGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: XaaTyrAspGlyPheGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: XaaTyrAspGlyGlyGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: XaaTyrAspGlyHisGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: XaaTyrAspGlyIleGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid /note=."alpha2(I) type I collagen" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: XaaTyrAspGlyLysGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: XaaTyrAspGlyLeuGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: XaaTyrAspGlyMetGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: XaaTyrAspGlyAsnGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: XaaTyrAspGlyProGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: XaaTyrAspGlyGlnGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: XaaTyrAspGlyArgGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: XaaTyrAspGlySerGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: XaaTyrAspGlyThrGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: XaaTyrAspGlyValGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: XaaTyrAspGlyTrpGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: XaaTyrAspGlyTyrGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: XaaTyrAspGlyArgGlyValAla 15 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: XaaTyrAspGlyGluGlyValLeu 15 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: XaaTyrAspGlyGluGlyVal 15 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: XaaTyrAspGlyGlu 15 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: XaaAspGlyGluGlyVal 15 (2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1, 5..6 (D) OTHER INFORMATION: Xaa in position 1 is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa in position 5 is cysteic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: XaaTyrAspGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: XaaTyrAspAlaGluGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: XaaTyrAspGlyGluAlaValGly 15 (2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: XaaTyrAspGlyGluGlyLeuGly 15 (2) INFORMATION FOR SEQ ID NO:30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: XaaTyrGluGlyGluGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: XaaTyrAspGlyAspGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: XaaTyrGluGlyAspGluValGly 15 (2) INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is Glu converted (by autoclaving the peptide) to pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: XaaTyrAspGlyGluGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is 2- aminoadipic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: XaaTyrAspGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: XaaTyrAspGlyGluGlyValVal 15 (2) INFORMATION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: XaaPheAspGlyGluGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: XaaTyrAspValGluGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Val is norvaline (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: XaaTyrAspGlyValGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: XaaTyrAspGlyGluGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: XaaTyrAspGlyGluGlyValGlyLeu 15 (2) INFORMATION FOR SEQ ID NO:41: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: GlyGlyTyrAspGlyGluGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: XaaTyrAspGlyGluGlyValGlyGlyLysLeu 1510 (2) INFORMATION FOR SEQ ID NO:43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: SerTyrAspGlyGluGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 8 (D) OTHER INFORMATION: Xaa is 1,2- diaminoacetaldehyde (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: XaaTyrAspGlyGluGlyValXaa 15 (2) INFORMATION FOR SEQ ID NO:45: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: GlyValGlyCysGlyAspTyrGln 15 (2) INFORMATION FOR SEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: XaaTyrAspGlyGluValValGly 15 (2) INFORMATION FOR SEQ ID NO:47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: XaaTyrAspGlyGluGlyGlyGly 15 (2) INFORMATION FOR SEQ ID NO:48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: XaaTyrAspGlyGluGlyAlaGly 15 (2) INFORMATION FOR SEQ ID NO:49: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: XaaTyrAspGlyGluGlyValAla 15 (2) INFORMATION FOR SEQ ID NO:50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: HisTyrAspGlyGluGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: ProTyrAspGlyGluGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: XaaTyrAspGlyGluCysGlyGly 15 (2) INFORMATION FOR SEQ ID NO:53: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: XaaValGlyGluGlyAspTyrAsn 15 (2) INFORMATION FOR SEQ ID NO:54: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: XaaTyrAspGlyGluGlyValPro 15 (2) INFORMATION FOR SEQ ID NO:55: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: XaaTyrAspGlyGluGlyIleGly 15 (2) INFORMATION FOR SEQ ID NO:56: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: XaaTyrAspGlyGluGlyValCys 15 (2) INFORMATION FOR SEQ ID NO:57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is 4Hyp (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: XaaTyrAspGlyGluGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:58: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is biotinylated Lys (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: XaaTyrAspGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:59: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is biotinylated Cys (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: XaaTyrAspGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:60: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 3 (D) OTHER INFORMATION: Xaa is a negatively charged amino acid residue (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is a negatively charged amino acid residue (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: XaaTyrXaaGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:61: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 3 (D) OTHER INFORMATION: Xaa is a negatively charged amino acid residue (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is Pro, Trp, Nva, or His (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: XaaTyrXaaGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:62: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 3 (D) OTHER INFORMATION: Xaa is a negatively charged amino acid residue (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is pyridinylalanine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: XaaTyrXaaGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:63: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 3 (D) OTHER INFORMATION: Xaa is a negatively charged amino acid residue (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is an alpha-amino acid residue bearing a thiol group (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: XaaTyrXaaGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:64: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 3 (D) OTHER INFORMATION: Xaa is a negatively charged amino acid residue (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is homocysteine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: XaaTyrXaaGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:65: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 3 (D) OTHER INFORMATION: Xaa is a negatively charged amino acid residue (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is an alpha-amino acid bearing a side chain amino or thiol group (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65: XaaTyrXaaGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:66: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 3 (D) OTHER INFORMATION: Xaa is a negatively charged amino acid residue (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is glutamine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: XaaTyrXaaGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:67: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 3 (D) OTHER INFORMATION: Xaa is a negatively charged amino acid residue (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is aspartic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: XaaTyrXaaGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:68: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 3 (D) OTHER INFORMATION: Xaa is a negatively charged amino acid residue (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is cysteic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: XaaTyrXaaGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:69: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 3 (D) OTHER INFORMATION: Xaa is a negatively charged amino acid residue (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is alpha- aminoadipic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: XaaTyrXaaGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:70: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 3 (D) OTHER INFORMATION: Xaa is a negatively charged amino acid residue (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is proline (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: XaaTyrXaaGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:71: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 3 (D) OTHER INFORMATION: Xaa is a negatively charged amino acid residue (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is tryptophan (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: XaaTyrXaaGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:72: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 3 (D) OTHER INFORMATION: Xaa is a negatively charged amino acid residue (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is norvaline (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: XaaTyrXaaGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:73: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 3 (D) OTHER INFORMATION: Xaa is a negatively charged amino acid residue (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is histidine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: XaaTyrXaaGlyXaaGlyValGly 15 (2) INFORMATION FOR SEQ ID NO:74: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa is pyroglutamic acid (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 3 (D) OTHER INFORMATION: Xaa is a negatively charged amino acid residue (ix) FEATURE: (A) NAME/KEY: misc-feature (B) LOCATION: 5 (D) OTHER INFORMATION: Xaa is histidine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: XaaTyrXaaGlyXaaGlyValGly 15 __________________________________________________________________________
Claims (12)
Gln-Tyr-Xaa-Gly-Xaa-Gly-Val-Gly
Priority Applications (1)
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US08/807,030 US5817755A (en) | 1995-05-19 | 1997-02-26 | Synthetic peptide analogs of cross-linked N-telopeptides of type I collagen |
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US08/446,206 US5750647A (en) | 1995-05-19 | 1995-05-19 | Synthetic peptide analogs of NTx |
PCT/US1996/007132 WO1996036645A1 (en) | 1995-05-19 | 1996-05-17 | Synthetic peptide analogs of cross-linked n-telopeptides of type i collagen |
US08/807,030 US5817755A (en) | 1995-05-19 | 1997-02-26 | Synthetic peptide analogs of cross-linked N-telopeptides of type I collagen |
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US08/446,206 Continuation-In-Part US5750647A (en) | 1995-05-19 | 1995-05-19 | Synthetic peptide analogs of NTx |
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US5817755A true US5817755A (en) | 1998-10-06 |
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US08/807,030 Expired - Lifetime US5817755A (en) | 1995-05-19 | 1997-02-26 | Synthetic peptide analogs of cross-linked N-telopeptides of type I collagen |
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US (2) | US5750647A (en) |
EP (1) | EP0827510A1 (en) |
JP (1) | JP3742434B2 (en) |
AU (1) | AU5796796A (en) |
WO (1) | WO1996036645A1 (en) |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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US6255056B1 (en) | 1998-06-19 | 2001-07-03 | Washington Research Foundation | Cartilage resorption assays |
EP0742902B1 (en) * | 1993-09-17 | 2001-11-21 | Osteometer Biotech AS | A method of assaying collagen fragments in body fluids, a test kit and means for carrying out the method and use of the method to diagnose the presence of disorders associated with the metabolism of collagen |
US6348320B1 (en) | 1998-06-19 | 2002-02-19 | Washington Research Foundation | Cartilage resorption assays measuring type II collagen fragments |
WO2002029409A2 (en) * | 2000-10-03 | 2002-04-11 | Rowett Research Institute | Method of assaying pyrrole-containing biological compounds |
US6602980B1 (en) | 1998-06-19 | 2003-08-05 | Washington Research Foundation | Collagen type III synthetic peptides for collagen resorption assays |
US20040048321A1 (en) * | 1998-06-19 | 2004-03-11 | Washington Research Foundation | Cartilage resorption assays |
US20050124071A1 (en) * | 2003-09-30 | 2005-06-09 | Kraus Virginia B. | Methods and compositions for diagnosing musculoskeletal, arthritic and joint disorders by biomarker dating |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
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US6110689A (en) * | 1994-01-21 | 2000-08-29 | Osteometer A/S | Method of assaying collagen fragments in body fluids, a test kit and means for carrying out the method and use of the method to diagnose the presence of disorders associated with the metabolism of collagen |
GB9617616D0 (en) | 1996-08-22 | 1996-10-02 | Osteometer Biotech As | Assaying protein fragments in body fluids |
DE69705423T2 (en) * | 1996-12-09 | 2002-05-16 | Osteometer Biotech As Herlev | SANDWEIGHT TEST FOR DETECTING COLLAGEN FRAGMENTS |
ES2214722T3 (en) * | 1997-07-31 | 2004-09-16 | Metra Biosystems, Inc. | METHOD OF TESTING PEPTIDES OF COLAGENO. |
US6197592B1 (en) * | 1999-07-15 | 2001-03-06 | Bio-Rad Laboratories, Inc. | Clinical control materials for detection of bone resorptive markers |
EA012656B1 (en) | 2005-05-25 | 2009-12-30 | Аэроджен, Инк. | Vibration systems and use thereof |
WO2023068248A1 (en) * | 2021-10-20 | 2023-04-27 | 積水メディカル株式会社 | Immunoassay method for cross-linked n-telopeptide of type i collagen, immunoassay kit, and antibody or antibody fragment thereof |
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- 1996-05-17 AU AU57967/96A patent/AU5796796A/en not_active Abandoned
- 1996-05-17 WO PCT/US1996/007132 patent/WO1996036645A1/en not_active Application Discontinuation
- 1996-05-17 EP EP96914682A patent/EP0827510A1/en not_active Ceased
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Also Published As
Publication number | Publication date |
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WO1996036645A1 (en) | 1996-11-21 |
JP3742434B2 (en) | 2006-02-01 |
US5750647A (en) | 1998-05-12 |
AU5796796A (en) | 1996-11-29 |
EP0827510A1 (en) | 1998-03-11 |
JPH11505804A (en) | 1999-05-25 |
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